The present invention relates to a method for extracting oleaginous substances from Ganoderma lucidum spores, which are germination-activated and epispore-broken. The extraction method involves the use of supercritical fluid carbon dioxide (xe2x80x9cSCFxe2x80x94CO2xe2x80x9d).
Ganoderma (Ganoderma lucidum Leyss ex Fr. Karst) is a polyporous fungus. It belongs to the class of Basidiomycetes, the family of Polypolaceae, and the genus of Ganoderma. In Chinese folklore, Ganoderma has been regarded as a panacea, which is probably due to certain efficacy of Ganoderma in treating many diseases. Some of the known medicinal or therapeutic effects of Ganoderma include treating patients with chronic bronchitis, chronic viral hepatitis, coronary heart disease, granulocytopenia, chronic Keshan disease, neurasthenia, progressive muscular dystrophy, atrophic myotonia and certain neurological diseases (See e.g., Liu et al., Chinese Medical Journal, 92:496-500 (1979)). There are also reports on Ganoderma as anti-HIV agent (See e.g., El-Mekkawy et al., Phytochemistry, 49: 1651-1657 (1998); Min et al., Chem. Pharm. Bull, 46: 1607-1612 (1998)), or for having anti-tumor, cardiovascular, antiviral, antibacterial, antiparasitic, and immune modulating activities (See e.g., Wasser et al., Critical Review in Immunology, 19:65-96 (1999)).
There are two major types of compounds found in Ganoderma which have been shown to be associated with the medicinal or therapeutic effects of Ganoderma. They are the polysaccharide compounds and the terpenoids. The polysaccharide compounds are primarily water-soluble. The terpenoids are oleaginous substances and are generally insoluble in water.
The polysaccharide compounds isolated from Ganoderma include hetero-xcex2-glucans and their protein complexes (such as xyloglucans and acidic xcex2-glucan-containing uronic acid, dietary fibers, lectins). The polysaccharides found in Ganoderma have been reported to possess anti-tumor and immune modulating effects (See Wasser et al., supra).
The Ganoderma terpenoids contain a lanostane skeleton. They are classified into several groups based on their carbon numbers and state of oxidation (Komoda et al., Chem. Pharm. Bull., 33:4829-4835 (1985)). These Ganoderma terpenoids include lanostanine-type triterpenoids (e.g., ganoderic acids A, B, C1, C2, D1, D2, E1, E2, F, G, H, I, J, K1, K2, L, Ma, Mb, Mc, Md, Me, Mf, Mg, Mi, Mj, Mk, Mn, N, O, P, Q, S, T, U, V, W, X, Y, and Z), 7-O-methyl-ganoderic acid O, trideacetyl ganoderic acid T, ganoderenic acids A, B, C, D, E, F, G, H, I, ganolucidic acids A, B, C, D, and E, lucidenic acids A, B, C, D1, D2, E1, E2, F, G, H, I, J, K, L, M, ganoderiol type 1 (A, B, F) and type 2 (C, D, E, F, G, H, and I), ganoderal A and B, epoxyganoderiol A, B, C, lucidone A, B, C, furanoganoderic acid, and other terpenoid components. Ganoderma terpenoids (e.g., ganoderic acids R, T, U-Z) have been reported to inhibit growth of hepatoma cells in vitro (See Toth et al., Tetrahedron Lett., 24:1081-1084 (1983)).
Ganoderma spores are tiny mist-like brown oval-shaped spores of (6xcx9c7) xcexcmxc3x97(10xcx9c12) xcexcm in sizes which are released at the pelius of mature Ganoderma lucidum. These spores contain the entire genetic materials and biological substances of Ganoderma. However, the wild Ganoderma spores are difficult to collect, particularly due to their short release period and low germination rate under unfavorable environmental conditions. Therefore, although it is known that the Ganoderma spores are of greater pharmaceutical values than the fruiting bodies of Ganoderma, due to difficulties associated with the collection of the Ganoderma spores, most of the studies on Ganoderma are conducted using the fruiting bodies of Ganoderma.
The biological substances within the Ganoderma spores which give rise to the therapeutic effects of Ganoderma are stored within the double-layered epispores of Ganoderma lucidum. However, these epispores have compact structure, which are extremely rigid and resilient. Therefore, it is very difficult to break-open the epispore layers of the Ganoderma spores and release the biological substances therein using conventional extraction methods.
There have been reports on methods for breaking the epispores of Ganoderma spores. For example, Japanese Patent No. JP52041208 discloses an extraction method for breaking Ganoderma spores using mechanical force. Chinese Patent No. CN1134306 teaches a method for breaking the sporoderm of the Ganoderma spores by soaking the spores in water, followed by microwave-heating. Chinese Patent No. CN1165032 teaches a method for breaking the cell wall of Ganoderma lucidum spores by digesting the spores with skin-dissolving enzymes such as lysozyme, snail enzyme, cellulase, or hemicellulase, followed by ultrasonic breakage of the cell walls at 20-50xc2x0 C. However, these methods use a mixed batch of spores collected from different stages of the Ganoderma lifecycle. It is known that the spores at different stages of the lifecycle produce different kinds and/or proportions of the biological substances, which may or may not possess the high level of therapeutic effects as expected. Therefore, the sporoderm-broken spores produced by these methods display inconsistent results and their respective medicinal effects vary.
There have also been reports on isolation or separation of the oleaginous substances (e.g., the terpenoids) from Ganoderma, most involving the use of organic solvents. For example, Min et al., Chem. Pharm. Bull., supra, disclose the isolation of lanostane-type triterpenes using column chromatography of a CHCl3-soluble fraction of the methanol extract of the Ganoderma spores. Lin et al., J. Chromatography, 410: 195-200 (1987) disclose the separation of oxygenated triterpenoids from Ganoderma lucidum by high-performance liquid chromatography of a methanolic extract of Ganoderma lucidum. These methods are unsatisfactory due to complex extraction procedures and low yield of the oleaginous substances.
In the present invention, a method for extracting the oleaginous substances from Ganoderma spores is provided. The Ganoderma spores to be used in the present invention are germination-activated to ensure that the biological substances are maximally produced. The epispores of the germination-activated Ganoderma spores are broken by a mechanical means to release the biological substances. Finally, the oleaginous substances of the biological substances are separated by a supercritical fluid carbon dioxide (SCFxe2x80x94CO2) extraction method. The present invention has the advantage of producing high yield of oleaginous substances from Ganoderma (i.e., the yield of the oleaginous substances is about 37% by weight of the entire biological substances released from Ganoderma). The oleaginous substances isolated based on the present method retain the special fragrance of Ganoderma and are without solvent residue and strange odor.
The present invention provides a method for extracting the oleaginous substances from the sporoderm-broken Ganoderma spores using a supercritical fluid carbon dioxide (SCFxe2x80x94CO2) extraction method. The Ganoderma spores are germination-activated and sporoderm-broken.
To obtain the germination-activated Ganoderma spores, the Ganoderma spores are first soaked in a nutritional solution which is suitable for inducing germination. The nutritional solution for germination purpose includes, but is not limited to, an immersed solution of Ganoderma fruiting body, a biotin solution, water, and an immersed solution of Ganoderma mycelium. The immersed solution of Ganoderma fruiting body or Ganoderma mycelium is preferably 0.5 to 25% by weight; the preferred biotin solution is 0.1 to 0.5% by weight. The ratio between the volume of the nutritional solution and the weight of Ganoderma spores is about 0.01 to 5 times. The preferred soaking time is between 10 minutes to 10 hours. The preferred soaking temperature is between 16xc2x0 C. and 43xc2x0 C.
The germination-induced Ganoderma spores are activated by placing the soaked Ganoderma spores in a well-ventilated culture box. The preferred relative humidity is 60% to 98%. The preferred temperature is 16xc2x0 C. to 43xc2x0 C. The preferred activation period is between 10 minutes and 24 hours.
The breakage of the epispores of the Ganoderma spores can be achieved by applying a mechanical means to the spores. The preferred mechanical means includes, without limitation, micronization, ultra-high-speed airstream, scissor-cut/grinding, and ultra-high pressure microstream. It is optional to incubate the germination-activated Ganoderma spores with enzymes such as chitinase and/or cellulase to soften the cell walls of the spores before applying the mechanical means to the spores.
The extraction of oleaginous substances from the germination activated and sporoderm-broken Ganoderma spores is carried out by a supercritical fluid carbon dioxide (SCFxe2x80x94CO2) extraction method. The method includes the steps of: (1) placing the spores in a pressure vessel; (2) contacting SCFxe2x80x94CO2 with the spores in the pressure vessel; and (3) depressurizing the pressure vessel to collect the oleaginous substances from the Ganoderma spores. The pressure in the pressure vessel is preferably between 5 M Psia (Pa) to 60 M Pa. The temperature in the pressure vessel is preferably maintained at 32xc2x0 C. to 85xc2x0 C. The preferred flow capacity rate of the pressure vessel is between 5 kg/h and 80 kg/h. The preferred extraction time is between 30 minutes and 6 hours.
Optionally, the sporoderm-broken Ganoderma spores are mixed with a carrier, such as water or 85% to 100% ethanol, before being placed in the pressure vessel. The preferred ratio of the carrier to the Ganoderma spores is 2% to 200% by weight. The oleaginous substances are preferred to be separated from the carrier by centrifugation.
The SCFxe2x80x94CO2 extraction method produces oleaginous substances from the Ganoderma spores, which are about 37% of the total weight of the Ganoderma spores. The oleaginous substances are transparent and contain a special fragrance of the Ganoderma spores. There is no trace of deposit, solvent residue, or oxidization in the oleaginous substances.
The oleaginous substances extracted from the sporoderm-broken Ganoderma spores possess medicinal effects, which include, without limitation, anti-tumor, anti-HIV or -HBV, and anti-immunological disorders. They can be used in treating patients with tumors, HIV or HBV infection, and immunological disorders.
The present invention provides an extraction method to isolate and separate the oleaginous substances from Ganoderma spores by using an SCFxe2x80x94CO2. The amount of the oleaginous substances produced by this method constituted about 37% of the total weight of the spores. In addition, the oleaginous substances produced by this method were well preserved in its natural state (i.e., free of solvent residue and strange odor). The strange odor is an indication that the oleaginous substances have been oxidized.
Conventionally, the ways to extract or separate substances in a mixture include distillation and solvent extraction. Distillation separates the substances in a mixture according to the boiling characteristics of each substance. Solvent extraction separates the substances in a mixture according to the hydrophilic and lipophilic property of each substance. Distillation cannot be used when the substances to be separated are thermally unstable. Solvent extraction has limited utility when the substances to be separated are so similar in solubility that efficient separation cannot be obtained.
Supercritical fluids (SCFs) technology is a viable alternative to the conventional extraction methods. SCFs are often referred to as dense gases. Technically, an SCF is a gas existing above its critical temperature and critical pressure. When a gas is compressed above its critical temperature, densities increase dramatically. Therefore, under a given set of conditions, an SCF may possess the density of a liquid while maintaining the diffusivity of a gas.
Each gas has a critical pressure (Pc) and a critical temperature (Tc), above each of which a supercritical fluid state is attained. Solvent properties of such SCFs have been found to be a complex function of the fluid density which in turn is a complex function of temperature and pressure. Thus, by varying the temperature and pressure of a supercritical fluid, extractions and precipitations can be carried out.
Carbon dioxide has proven to be a particularly advantageous gas to use in SCF extractions because it possesses good solvent properties and has low chemical reactivity and toxicity. In addition, carbon dioxide is not flammable, is inexpensive and may be readily recycled, and leaves no undesirable residues in the precipitates. Carbon dioxide has a Pc of 73.8 bar, a Tc of 31.1xc2x0 C., and a density at the Pc and Tc of 0.468 g/cc.
By use of SCFxe2x80x94CO2, any material in a mixture which exists in or which can be converted to a physical state that is permeable to the carbon dioxide under supercritical conditions will be dissolved in the carbon dioxide and be separated from the mixture. The principle behind the SCFxe2x80x94CO2 extraction method is that under the high pressures required for extraction with gases in the supercritical fluid, the solubility of many organic compounds is increased. This, combined with the greater diffusivity of supercritical fluid over conventional solvents, results in a more rapid mass transfer through the material to be extracted, and thus a faster rate of extraction. Supercritical fluid gases have the ability to selectively dissolve and extract organic species from organic mixtures, organic/aqueous mixtures, organic/inorganic matrices, and lipophilic/hydrophilic matrices. Theoretically, the higher the pressure, the greater the efficiency of the extraction. Essentially, most or all of the oleaginous substances (with low solubility in water) in the Ganoderma spores should be dissolved in the carbon dioxide.
The tiny spores of Ganoderma lucidum has an extremely hard and resilient, double-layered epispore. In the wild, the germination of the spores of Ganoderma lucidum is relatively slow and their germination rate is extremely low. It takes about 24 to 48 hours for the germ tubes of the spores start to sprout under proper conditions, and the capillitia start to form branches after 72 hours, with a germination rate of only 3-15%.
When the tiny spores of Ganoderma lucidum were extracted under SCFxe2x80x94CO2, only approximately 3.5% of the oleaginous substances were recovered.
In order to maximize the production of oleaginous substances from Ganoderma lucidum, a germination-activation procedure was designed. This procedure was followed by a sporoderm-breaking process to break open the cell walls of the Ganoderma epispores. Finally, the germination-activated, sporoderm-broken Ganoderma spores were extracted under the SCFxe2x80x94CO2 to separate the oleaginous substances from the spores.
It was noted that when the dormant Ganoderma spores were germination-activated, the production rate of the biological substances in the spores reached the maximum. These biological substances contain, inter alia, active genes and promoters, active enzymes, sterols, cytokines, interferons, lactone A, ganoderma acid A, triterpenes, polysaccharides, vitamins, superoxide dismutases (SOD), glycoproteins, etc. These biological substances demonstrate superb medicinal effects, particularly on stimulating and modulating the nervous system and the immune system. Also, it was noted that during the germination-activation period, the resilience of the epispore significantly decreased, which in turn increased the penetration rate of the cell walls of the epispore.
The results of the animal and clinical studies on the effect of germination-activation showed that the biological substances produced from the germination-activated Ganoderma spores demonstrated inhibitory effects on liver cancer by suppressing the activity of telomerase in the hepatic cancerous tissue. These biological substances also demonstrated therapeutic effects on HBV infection. Additionally, when the germination-activated Ganoderma spores were given to animals, the results showed that the sporoderm-unbroken spores had an anti-tumor rate of 23.2%, which was substantially lower than the sporoderm-broken spores, which had an anti-tumor rate of 86.1%.
The extraction method of the present invention is described as follows:
1. Collection of Ganoderma Spores. Mature and plump Ganoderma spores were collected at the appropriate release time from Ganoderma lucidum cultured on log. It was advantageous to culture Ganoderma on log, because the spores thus produced were fresher and more nutritious and the penetration/ breaking rate for the epispores was much higher.
2. Induction of Ganoderma Spores Germination. The selected spores were soaked in a nutritional solution which could be distilled water, a saline solution, a solution which had been immersed with the fruiting bodies of Ganoderma or the mycelia of Ganoderma. The purpose of soaking the spores in the nutritional solution was to enable and accelerate the germination of the spores. Examples of the nutritional solution include 0.5xcx9c25% by weight of the immersion solution of the Ganoderma fruiting bodies or mycelia, 0.1xcx9c0.5% by weight of the biotin solution, etc. The nutritional solution was about 0.01xcx9c5 times of the weight of the Ganoderma spores. The soaking time was about 10 minutesxcx9c8 hours. The temperature was about 16xcx9c43xc2x0 C.
3. Activation of the Germinated Ganoderma Spores. To activate the germinated Ganoderma spores, the soaked spores were removed from the nutritional solution and excess solution was allowed to drip. The soaked spores were then placed in a well-ventilated culture box which was kept in constant temperature and humidity. The relative humidity in the culture box was maintained at about 60xcx9c98%. The temperature of the culture box was maintained at about 16xcx9c48xc2x0 C. The time for activating the spores was about 10 minutesxcx9c24 hours.
4. Penetration/Breakage of the Epispores. After the Ganoderma spores were germination-activated, the spores were further broken by a mechanical means. Examples of the mechanical means used to break the spores include micronization, roll-pressing, or scissor-cut/grinding, microstream-impact crushing, ultra-high-speed airstream impact crushing, ultra-high pressure microstream crushing, ultra-low temperature crushing etc.
Before breaking the epispores, it was optional to treat the spores with enzymes such as chitinase and cellulase to soften the cell walls of the epispores. The enzyme-treated spores could be separated from the reaction mixture by centrifugation at about 3,000xcx9c30,000 rpm or ultra-filtration using a filter with about 10,000 molecular weight cut-off.
5. Extraction of Oleaginous Substances with SCFxe2x80x94CO2. The extraction of the oleaginous substances from the sporoderm-broken spores was conducted in an SCFxe2x80x94CO2 extracting apparatus, which included a CO2 source, a compressor, a heat exchanger, a pressure regulator, and a pressure vessel. Alternatively, any conventional supercritical fluid extraction equipment which contains an extractor (i.e., the pressure vessel) and a separator would also be suitable for the extraction. To operate, the sporoderm-broken spores were placed in the pressure vessel. The carbon dioxide was flowed through the compressor and heat exchanger to achieve greater than supercritical temperature and pressure, and then flowed through the spores in the pressure vessel. The SCF was then removed from the pressure vessel and depressurized to evaporate the carbon dioxide. The supercritical pressure used in the present method was about 5xcx9c60 M Pa. The supercritical temperature was in the present method was about 32xcx9c85xc2x0 C. The flow volume rate of CO2 was about 5xcx9c80 kg/h. The extraction time was about 0.5xcx9c6 hours.
It was optional to add a carrier to the spores between initiating the SCFxe2x80x94CO2 extraction. Examples of the carrier include water or 85xcx9c100% of ethanol. The ratio of the carrier to the spores was about 2xcx9c200% (v/w). When the carrier was added to the spores, the oleaginous substances could be separated from the rest of the spores by centrifugation at about 3,000xcx9c30,000 rpm.